Institute of Sciences of Food Production (ISPA), National Research Council of Italy (CNR), Bari, Italy.
Anal Bioanal Chem. 2011 Nov;401(9):2831-41. doi: 10.1007/s00216-011-5354-z. Epub 2011 Sep 4.
Humans and animals can be simultaneously exposed through the diet to different mycotoxins, including aflatoxins, ochratoxin A, deoxynivalenol, zearalenone, and fumonisins, which are the most important. Evaluation of the frequency and levels of human and animal exposure to these mycotoxins can be performed by measuring the levels of the relevant biomarkers in urine. Available data on the toxicokinetics of these mycotoxins in animals suggest that aflatoxin M(1) (AFM(1)), ochratoxin A (OTA), deoxynivalenol (DON)/de-epoxydeoxynivalenol (DOM-1), alpha-zearalenol (α-ZOL)/beta-zearalenol (β-ZOL), and fumonisin B(1) (FB(1)) can be used as urinary biomarkers. A liquid chromatographic-tandem mass spectrometric method has been developed for simultaneous determination of these mycotoxin biomarkers in human or animal urine. Urine samples were purified and concentrated by a double cleanup approach, using a multitoxin immunoaffinity column and a reversed-phase SPE Oasis HLB column. Separation of the biomarkers was performed by reversed-phase chromatography using a multi-step linear methanol-water gradient containing 0.5% acetic acid as mobile phase. Detection and quantification of the biomarkers were performed by triple quadrupole mass spectrometry (LC-ESI-MS/MS). The clean-up conditions were optimised to obtain maximum analyte recovery and high sensitivity. Recovery from spiked samples was performed at four levels in the range 0.03-12 ng mL(-1), using matrix-matched calibration curves for quantification. Mean recoveries of the biomarkers tested ranged from 62 to 96% with relative standard deviations of 3-20%. Enzymatic digestion with β-glucuronidase/sulfatase resulted in increased concentrations of the biomarkers, in both human and pig urine, in most samples containing measurable concentrations of DON, DOM-1, OTA, α-ZOL, or β-ZOL. A highly variable increase was observed between individuals. Co-occurrence of OTA and DON in human urine is reported herein for the first time.
人类和动物可通过饮食同时暴露于不同的真菌毒素，包括黄曲霉毒素、赭曲霉毒素 A、脱氧雪腐镰刀菌烯醇、玉米赤霉烯酮和伏马菌素，这些毒素是最重要的。可通过测量尿液中相关生物标志物的水平来评估人和动物接触这些真菌毒素的频率和水平。关于这些真菌毒素在动物体内的毒代动力学的现有数据表明，黄曲霉毒素 M1（AFM1）、赭曲霉毒素 A（OTA）、脱氧雪腐镰刀菌烯醇（DON）/去环氧脱氧雪腐镰刀菌烯醇（DOM-1）、α-玉米赤霉烯醇（α-ZOL）/β-玉米赤霉烯醇（β-ZOL）和伏马菌素 B1（FB1）可用作尿液生物标志物。已经开发了一种液相色谱-串联质谱法，用于同时测定人或动物尿液中的这些真菌毒素生物标志物。通过使用多毒素免疫亲和柱和反相 SPE Oasis HLB 柱的双重净化方法，对尿液样品进行净化和浓缩。使用含有 0.5%乙酸的甲醇-水多步线性梯度作为流动相，通过反相色谱法分离生物标志物。通过三重四极杆质谱（LC-ESI-MS/MS）进行生物标志物的检测和定量。优化了净化条件以获得最大的分析物回收率和高灵敏度。使用基质匹配校准曲线进行定量，在 0.03-12ng/mL 的范围内，在 4 个浓度水平上进行加标样品的回收率测定。所测试的生物标志物的平均回收率范围为 62%至 96%，相对标准偏差为 3%至 20%。在大多数含有可测量浓度的 DON、DOM-1、OTA、α-ZOL 或β-ZOL 的样品中，用β-葡萄糖醛酸酶/硫酸酯酶进行酶消化后，生物标志物的浓度增加，在人和猪的尿液中均如此。个体之间观察到高度可变的增加。本文首次报道了人尿中 OTA 和 DON 的共存。
